1. Field of the Invention
The present invention relates to a process for the preparation of antitumour agents The present invention particularly relates to a process for the preparation of novel pyrrolo[2,1-c][1,4]benzodiazepines useful as potential antitumour agents. More particularly, the present invention relates to a process for the preparation of 8-methoxy-7-(3-[7-methoxy-5-oxo(11aS)-2,3,4,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4][benzodiazepine-8-yloxyalkyloxy)-(11aR)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione, with aliphatic chain length variations and their 2-hydroxy and 2-acetyloxy derivatives.
2. Description of Related Art
In the past few years, a growing interest has been shown in the development of new pyrrolo[2,1-c][1,4]benzodiazepines (PBDs). These antibiotics react covalently with DNA to form an N2-guanine adduct that lies within the minor groove of duplex DNA via an acid-labile animal bond to the electrophilic imine at the N10-C11 position. (Ref. Kunimoto, S., Masuda. T., Kanbayshi, N., Hamada. M., Naganawa. H., Miyamoto, M., Takeuchi, T. and Unezawa, H., J. Antibiot., 1980, 33, 665; Kohn K. W. and Speous, C. L., J. Mol. Biol., 1970, 51, 551.; Hurley, L. H., Gairpla, C. and Zmijewski, M., Biocheim. Biophys Acta., 1977, 475, 521.; Kaplan D. J. and Hurley, L. H. Biochmestry, 1981, 20, 7572). The molecules have a right-handed twist allowing them to follow the curvature of the minor groove of B-form double-stranded DNA spanning three base pairs. A recent development has been the linking of two PBD units through their C-8 positions to give bisfunctional alkylating agents capable of cross-linking DNA (Ref. Thurston, D. E., Bose, D. S., Thomson, A. S., Howard, P. W., Leoni, A., Croker, S. J., Jenkins, T. C., Neidle, S. and Hurley. L. H., J. Org. Chem., 1996, 61, 8141-8147).
Naturally occurring pyrrolo[2,1-c][1,4]benzodiazepines belong to a group of antitumour antibiotics derived from Streptomyces species. Recently, emphasis has been given to PBD systems as they can recognize and bind to a specific sequence of DNA. Examples of naturally occurring PBD""s include anthramycin, tomaymycin, sibiromycin and neothramycin. 
In the last decade a number of PBD""s, particularly C-8 linked PBD dimers, have been designed and synthesized to explore their effectiveness as DNA-sequence selective agents (Ref: Bose, D. S., Thomson, A. S., Ching, J. A., Hartley, J. A., Berardini. M. D., Jenkins. T. C., Neidle, S., Hurley, L. H. and Thurston. D. E., J. Am. Chem. Soc. 1992, 114, 4939).
Pyrrolo[2,1-c][1,4]benzodiazepine-5,11-diones are a class of compounds that bind to DNA by non-covalent interactions such as hydrophobic, Vanderwalls interactions and hydrogen bonding between ring substituents and DNA, and are also responsible for influencing sequence selectivity. Some dilactams such as (7-methoxy-2-methylcarbonyloxy-5,11-dioxo-(2S)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]-benzodiazepine-5,11-dione-8-yl acetate is reported to possess significant in vivo antitumour activity in P388 rat model. (Ref.: Kaneko, T., Wong, H., Doyle, T. W., Rose, W. C and Bradner. W. T., J. Med Chem. 1985, 28, 388)
The main object of present invention is to provide a new class of C-8 linked PBD dimers, wherein one PBD has an imine functionality while the other has an amide group. It has been envisaged that such a mixed dimer could offer more insight not only for the covalent binding but also the role played by non-covalent interactions with DNA bases.
Another object of the invention is to provide a process for the preparation of novel Pyrrolo[2,1-c][1,4]benzodiazepines useful as antitumour agents.
Accordingly, the present invention provides a process for the preparation of a Pyrrolo[2,1-c][1,4]benzodiazepine of formula VI wherein R is H, OH, OAc; R1, is H; and n is 3 to 5. 
The process comprises:
reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzyl]-pyrrolidine-2-carboxy-carbaldehyde diethyl thioacetal of formula I wherein R1 is H 
with a dibromoalkane in an aprotic water miscible organic solvent in the presence of a mild inorganic base with a refluxing temperature for a period upto 48 hours; isolating (2S)-N [4-(3-bromoalkoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II wherein R1 is H; 
reacting the compound of formula II with a dilactam of formula III wherein R is H, OH or OAc 
in the presence of a mild inorganic base in the presence of an aprotic water miscible organic solvent with a refluxing temperature for a period upto 48 hours;
isolating 8-([(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal)-alkoxy-7-methoxy-2,3,5,10,11,11a-hydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione of formula IV wherein R is H, OH or OAc; R1 is H; and n is 3 to 5; 
reducing the above nitro compound of formula IV with SnCl22H2O in the presence of an organic solvent with a reflux temperature, isolating the 8-([(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal)-alkoxy-7-methoxy-2,3,5,10,11,11a-hydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione of formula V, wherein R is H, OH or OAc; R1, is H; and n is 3 to 5; and 
reacting the amino compound of formula V with a deprotecting agent to obtain the pyrrolo[2,1-c][1,4]benzodiazepines of formula VI wherein R, R1 and n are as stated above.
In one embodiment of the invention, the aprotic water miscible organic solvent used is selected from the group consisting of acetone, tetrahydrofuran (THF) and dimethylformamide (DMF).
In another embodiment of the invention the mild inorganic base used for the reaction of compound of formula I is selected from the group consisting of K2CO3, BaCO3, Na2CO3, and mixtures thereof, and the reaction is refluxed at a temperature for a period in the range of 24 to 48 hours.
In another embodiment of the invention, the compound of formula II comprises (2S)-N[4-(3-bromopropoxy)-5-methoxy-2-nitrobenzyol]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal, (2S)-N-[4-(4-bromobutoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal and/or (2s)-N-[4-(5-bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal wherein R1 is H.
In another embodiment of the invention, the aprotic water miscible organic solvent used when reacting the compound of formula II with the debenzylated dilactam of formula III is selected from acetone, THF and DMF. (Ref: I. Kaneko, E.: Wang, H.; Doyle T. W. The J. Antibiotics. 1984, 3, 300).
In yet another embodiment of the invention, the compound of formula IV comprises 8-([(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal)-propoxy-7-methoxy-2,3,5,10,11,11a -hydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione, 8-([(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal)-butoxy-7-methoxy-2,3,5,10,11,11a-hydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione, and/or 8-[[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-pentyloxy-7-methoxy-2,3,5,10,11,11a-hydro-1H-pyrrolo[2,1,-c][1,4]benzodiazepine-5,11-dione, wherein R is H, OH or OAc and R1 is H.
In another embodiment of the invention, the organic solvent used in the reduction of the nitrothioacetal compounds of the formula IV with SnCI2.2H2O is selected from the group consisting of MeOH, DMF, 1,4-dioxane, and mixtures thereof.
In a further embodiment of the invention, the compound of formula V obtained by the reduction of compound of formula IV comprises 8-[[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-propoxy-7-methoxy-2,3,5,10,11,11a-hydro-1H-pyrrolo[2,1,-c][1,4]benzodiazepine-5,11-dione,8-[[(2S)-N-5-(methoxy 2-aminobenzyol]pyrrolidin-2-carbaldehyde diethylthioacetal]-butoxy-7-methoxy-2,3,5,10,11,11a-hydro-1H-pyrrolo[2,1,-c][1,4]benzodiazepine-5,11-dione, and/or 8-[[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-pentyloxy-7-methoxy-2,3,5,10,11,11a-hydro-1H-pyrrolo[2,1,-c][1,4]benzodiazepine-5,11-dione, wherein R is H, OH or OAc and R1 is H.
In another embodiment of the invention, the deprotecting agent is selected from the group consisting of HgCl2/HgO, HgCl2/CaCO3 and mixtures thereof.
In another embodiment of the invention, the organic solvent used in the reduction of compound of formula V to obtain the compound of formula VI is selected from acetonitrile and MeOH.
In a further embodiment of the invention, the compound of formula IV is reduced with SnCl22H2O in the presence of an organic solvent with a reflux temperature to obtain compound of formula IV comprising 8-[[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-propoxy-7-methoxy-2,3,5,10,11,11a-hydro-1H-pyrrolo[2,1,-c][1,4]benzodiazepine-5,11-dione, 8-[[(2S)-N-5-methoxy-2-aminobenzyol]pyrrolidin-2-carbaldehyde diethylthioacetal]-butoxy-7-methoxy-2,3,5,10,11,11a-hydro-1H-pyrrolo[2,1,-c][1,4]benzodiazepine-5,11-dione, 8-[[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-pentyloxy-7-methoxy-2,3,5,10,11,11a-hydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione, wherein R is H, OH or OAc and R1 is H. The compound of the formula V is reacted with a deprotecting agent selected from HgCl2/HgO and HgCl2/CaCO3 in the presence of an organic solvent selected from acetonitrile and MeOH, and 8-methoxy-7-3-[7-methoxy-5-oxo-(11aS)-2,3,4,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxypropoxy-(11aR)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione, 8-methoxy-7-4[7-methoxy-5-oxo-(11aS)-2,3,4,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxybutoxy-(11aR)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5-11-dione, 8-methoxy-7-5-[7 methoxy-5-oxo-(11aS)-2,3,4,11a-tetrahydro-1H-pyrrolo[2,1-c]1,4]benzodiazepine-8-yloxypentyloxy-(11aR)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione of the formula VI wherein R, R1 and n are as stated above, are recovered from the reaction mixture.
The precursor, (2S)-N-(4-hyxroxy-2-methoxy-2-nitrobenzoyl) pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula I (intermediates of DC-81) is prepared by literature methods (Ref: Thurston, D. E.; Murthy, V. S. Langley, D. R.; Jones, G. B. Synthesis, 1990, 81).
Some representative compounds of formula VI are given below:
8-methoxy-7-3-[7-methoxy-5-oxo-(11aS)-2,3,4,11a-tetrahydro-1H-pyrrolo [2,1-c][1,4]benzodiazepine-8-yloxy]propoxy-(11aR)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione;
8-methoxy-7-3-[7-methoxy-5-oxo-(11aS)-2,3,4,11a tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy]butoxy-(11aR)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione;
8-methoxy-7-3-[7-methoxy-5-oxo-(11aS)-1,2,3,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]-benzodiazepine-8-yloxy]pentyloxy-(11aR)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11 dione;
2-hydroxy-8-methoxy-7-3-[7-methoxy-5-oxo-(11aS)-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy]propoxy-(2R,11aS)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c[1,4]benzodiazepine-5,11-dione;
2-hydroxy-8-methoxy-7-5-[7-methoxy-5-oxo(11aS)-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy]butoxy-(2R,11aS)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione;
2-hydroxy-8 methoxy-7-5-[7-methoxy-5-oxo(11aS)-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy]pentryloxy (2R,11aS)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione;
8-methoxy-7-3[7-methoxy-5-oxo-(11aS)-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-pine-8-yloxy]propoxy)-5,11-dioxo-(2R,11aR)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-2-yl acetate;
8-methoxy-7-4-[7-methoxy-5-oxo-(11aS)-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy]butoxy)-5,11-diozo-(2R,11aR)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-2-yl acetate; and
8-methoxy-7-(5-(7-methoxy-5-oxo-(11aS)-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)pentyloxy)-5,11-dioxo-(2R,11aR)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-2-yl acetate.
The molecules synthesized are of immense biological significance with potential sequence selective DNA-binding property. This [resulted] results in design and synthesis of new congeners as illustrated in Scheme-1, having:
1. Ether linkage at C-8 position of DC-81 intermediates with dilactams.
2. Refluxing the reaction mixture for 24-48 h.
3. Synthesis of C-8 linked PBD antitumour antibiotic imines.
4. Purification by column chromatography using different solvents like ethylacetate, hexane, dichloromethane and methanol.
The reaction mechanism for the preparation of the product of the invention is given below: 